The establishment of many novel bioluminescent enzymes (luciferases) has recently been reported. For example, Promega reports the establishment of a novel luminescent enzyme originating from a deep-sea shrimp (Non-patent Document 1). The molecular weight of this enzyme is half (19 kD) that of a known Renilla luciferase (RLuc), and its luminescence intensity is 100 folds greater. Further, Shigeri et al. reports 11 types of plankton-derived bioluminescent enzyme (Non-patent Document 20). Some of these luminescent enzymes were evaluated to have a luminescence intensity comparable to that of RLuc.
Further, some deep-sea luminescent animals belonging to the Augaptiloidea superfamily have heretofore been discovered (Non-patent Document 2). In addition, a luminescent enzyme originating from Gaussia princeps (GLuc), a luminescent enzyme originating from Metridia longa (MLuc), and luminescent enzymes originating from Metridia pacifica (MpLuc1 and MpLuc2), which all belong to the Metridinidae family, have also been discovered (Non-patent Documents 15, 19, and 21).
Meanwhile, research for improving the luminescence intensity or luminescence stability of these luminescent enzymes has progressed. Loening et al. established a stable RLuc variant having high luminescence intensity by using a method of introducing amino acid mutations into RLuc (Non-patent Document 14). In this study, a “consensus sequence-driven mutagenesis strategy” was used to specify the mutation introduction site (Non-patent Document 13). Further, the present researchers also succeeded in increasing the luminescence intensity and luminescence stability of GLuc, MpLuc1, and MLuc, which are luminescent enzymes originating from deep-sea luminescent animals, by using a method of predicting the enzyme active site based on a hydrophilic amino acid distribution chart, and introducing a variant into the site (Non-patent Document 11). However, these luminescent enzymes were still insufficient as enzymes usable for various bioassays, and improvement in luminescence characteristics, such as increase in luminescence intensity, has been desired.
The present inventors once suggested production of a thermodynamically stable luminescent enzyme sequence with an attempt to obtain information regarding the luminescence characteristics by bisecting a single luminescent enzyme sequence, and aligning the first half and the second half of the sequence based on the similarity in amino acid (single sequence alignment; SSA) (Non-patent Document 3). This method is based on the premise that a marine animal-derived luminescent enzyme has two enzyme active sites. By aligning the two enzyme active sites based on similar amino acids, it is possible to easily examine the similarity of the enzyme active site in the first half and the enzyme active site in the second half. This method attempts to produce a thermodynamically stable luminescent enzyme sequence by increasing the similarity of the first and second half of the sequence on the aforementioned presumption that the amino acid frequency is relevant to the thermodynamic stability.
Meanwhile, various applied technologies using a bioluminescent enzyme as a “reporter” have also been developed. Niu et al. classified the bioassays using a bioluminescent enzyme as a reporter into three groups: “basic,” “inducible,” and “activatable” (Non-patent Document 16). This classification is based on the characteristics of the reporter gene. First of all, the difference between “basic” and “inducible” is the presence or absence of an expression controlling character in the reporter expression by the promoter, and the difference in expression amount. A later-described antibody having a bioluminescent enzyme attached thereto corresponds to “basic,” and the bioluminescence resonance energy transfer (BRET) and two-hybrid assay belong to the category of “inducible.” The reporter-gene probes, which belong to the category of “activatable,” are characterized in that the reporter actively responds to ligand stimulation and produces bioluminescence. The later-described protein complementation assay (PCA), protein splicing assay (PSA), integrated-molecule-format bioluminescent probe, bioluminescent capsule, and the like belong to the category of “activatable.”
For the bioassays (hereinafter may also be simply referred to as “reporter assays”) using these bioluminescent enzymes as a reporter, various luminescent probes have been actively developed based on the aforementioned novel luminescent enzymes. The present inventors have heretofore conducted research and development regarding bioluminescence imaging using unique molecular design technology. More specifically, the inventors developed a method of measuring translocation of transcription factors into the nucleus or nongenomic protein-protein interactions in the cytosol using protein splicing (Non-patent Documents 7 and 8), and an integrated-molecule-format bioluminescent probe in which all of the necessary elements for signal recognition and bioluminescence emission are integrated (Non-patent Documents 4 and 6). Thereafter, the probes were multicolorized, and developed to be capable of simultaneous imaging of multiple signal-transduction processes (Non-patent Document 12). Moreover, the inventors further developed a circular permutation technique (Non-patent Document 9) and a molecular design technology using low-molecular-weight bioluminescent enzymes (Non-patent Document 12) as strategies for improving the ligand sensitivity of the bioluminescent probe. These technologies have been used as means for efficiently measuring molecular phenomena in cellular and cell-free systems.
Regarding the main research tools for exploring intra- or extracellular molecular phenomena, fluorescence imaging has been used more widely than luminescence imaging. However, due to their autofluorescence property, fluorescent proteins generate a high background, requiring an external light source. Therefore, fluorescence imaging requires a large instrumentation, such as a fluorescence microscope, and a sophisticated light-filtering system. Fluorescence imaging also has a drawback in that the maturation of a fluorescence chromophore takes at least several hours to several days. Further, since the number of simultaneously observable cells is limited for each measurement with a fluorescence microscope, quantitative measurement has been problematic (Non-patent Document 8).
On the other hand, bioluminescence imaging using a bioluminescent enzyme has, despite its many advantages, a critical problem regarding poor luminescence intensity of bioluminescent enzymes. This problem has decreased the popular use of bioluminescence imaging, compared with fluorescence imaging. Because of this poor bioluminescence intensity of bioluminescent enzymes, high-sensitivity detectors were required; therefore, bioluminescent enzymes have been considered inappropriate for single-cell imaging or exploration of organelles.
Further, studies on multicolor fluorescent proteins have greatly progressed, and many facts regarding their coloring mechanisms have been discovered; thus, many fluorescent proteins with diversified fluorescent characters have been developed based on these study results. In contrast, only limited kinds of bioluminescent enzymes exhibit multiple colors. Although it has been known that diversification of bioluminescent colors is advantageous in that (i) it enables simultaneous measurement of multiple cellular signals, and that (ii) it ensures a tissue permeability of red-shifted bioluminescence in living subjects, nearly no systematic study for diversifying the colors of bioluminescent enzymes based on their luminescence mechanisms has been conducted.
Accordingly, there has been a strong desire to newly establish a high-performance bioluminescent enzyme, increase its luminescence intensity and stability, and ensure heat resistance and salt tolerance. Further, a systematic study for red-shift of wavelength of bioluminescent color has also been highly desired.
In addition, appropriate selection of the reaction solution is an important factor in bioassays, and may influence the assay results. In particular, (1) reporter-gene assay, (2) two-hybrid assay, (3) enzyme-linked immunosorbent assay, and (4) radioimmunoassay (RIA) (Non-patent Document 22 and Non-patent Document 23) require more careful selection of the reaction solution.
A bioassay using a so-called “molecular probe” is another example of a bioassay in which the results greatly depend on the selection of the reaction solution. For example, color imaging of intracellular protein-protein interaction, i.e., (1) FRET assay using fluorescence resonance energy transfer of fluorescent protein (Non-patent Document 24), (2) 2-molecule-format protein complementation assay (PCA) characterized by luminescence recovery by bisection of a fluorescent protein or a luminescent protein into two fragments and recombining the fragments, and the like have been developed (Non-patent Document 25). The present inventors also developed an assay method using a protein splicing reaction (protein splicing assay (PSA)) (Non-patent Document 8).
Thereafter, the present inventors developed (1) an integrated-molecule-format bioluminescent probe (may also be simply referred to as a single-chain probe) that enables detection of a single fusion molecular protein-protein interaction (Non-patent Document 6 and Patent Document 4), and, as a derivative method thereof, (2) a bioluminescent probe that is produced through circular permutation of the gene sequence of a luminescent enzyme (Non-patent Document 8 and Patent Document 3). The present inventors further developed (3) a molecular stress sensor (molecular tension-indexed bioluminescent probe) based on the difference in enzymatic activity caused by an artificial stress applied to a luminescent enzyme (Non-patent Document 26 and Patent Document 5).
Recently, the present inventors developed a multiple recognition-type bioluminescent probe obtained as a combined technique of reporter-gene assay and integrated-molecule-format bioluminescent probe (Non-patent Document 27). This probe is characterized by two sensing steps for a single target substance. The present inventors further developed a multicolor bioluminescence imaging probe set by combining two colors of integrated-molecule-format bioluminescent probes (Patent Document 25). This probe is characterized by multicolor imaging of multiple aspects of bioactivity of a test substance.
Bioassays indispensably require a reaction solution, and are roughly classified into (1) a method using a fluorescent protein and (2) a method using a bioluminescent enzyme (luciferase), depending on the type of the luminescence signal. In the method using a fluorescent protein, a high background is generated due to the autofluorescence, and an external light source is necessary. Further, a relatively large luminescence detector having a precise spectral filter is problematically necessary to measure the fluorescence (e.g., a fluorescence microscope) (Non-patent Document 8). On the other hand, although the method using a bioluminescent enzyme does not have the above problems, it indispensably requires substrates because of a drawback such that the light emission of bioluminescence is weaker than that of fluorescence. Further, since the method using a bioluminescent enzyme relies on the luminescence of enzyme, easy changes in luminescence quantity depending on the salt concentration, temperature, pH, heavy-metal ion concentration, and the like become problematic. The method using fluorescence also has similar problems. Therefore, to fix the pH and optimize the luminescence reaction conditions, reaction solutions are widely used both in the fluorescence method and the luminescence method.
Considering such circumstances, the determination of the optimal buffer condition is an important factor to accomplish a successful assay in the various known assays using fluorescence or bioluminescence. Further, optimization of the reaction solution and the additives according to the characteristics of the bioassay has been desired so as to obtain sufficient detection sensitivity, selectivity, and signal stability.
To improve the assay effect, various additives have been used for reaction solutions (assay buffer). The additives must have functions for ensuring homogenous assay conditions, including (1) prevention of protein decomposition by protease, (2) suppression of influences of interfering substances, (3) ensuring the function as a buffer solution for supporting stable signal generation, and (4) causing mild breakage of the plasma membrane. Therefore, the additives (5) must stabilize the protein and (6) must not inhibit the probe performance that is the core of the luminescence reaction.
The major additives of the reaction solution include, as salts, NaCl, KCl, (NH4)2SO4, and the like; as an SH reagent, mercapto ethanol, DTT, and the like; as a polyol, glycerol, sucrose, and the like; and as a chelating reagent, EGTA, EDTA, and the like.
Examples of surfactants include polyoxyethylene (10) octylphenyl ether (Triton X-100; TX100), Nonidet P-40 (NP40), polyoxyethylene sorbitan monolaurate (Tween 20; TW20), polyoxyethylene sorbitan monooleate (Tween 80; TW80), polyoxyethylene (20) cetyl ether (Brij58), sodium dodecyl sulfate (SDS), and the like. Heretofore, a suitable surfactant has been selected by referring to the order of the hydrophilic degree of the surfactants, which is TW20>Brij58>TW80>TX100>NP40, and the order of the degree of surface activity, which is NP40>TX100>Brij58>TW20>TW80.
Examples of protease inhibitors to be used for inhibiting protein decomposition include aprotinin (molecular weight: 6.5 kD), leupeptin (molecular weight: 427), pepstatin A (molecular weight: 686), phenylmethylsulfonyl fluoride (PMSF, molecular weight: 174), antipain (molecular weight: 605), chymostatin (molecular weight: 608) and the like. Further, Pefabloc SC (AEBSF, 240 Da), DFP (184 Da), p-APMSF (216 Da), STI (20,100 Da), leupeptin (460 Da), N-tosyl-L-phenylalaninechloromethylketone, 3,4-dichloroisocoumarin (215 Da), EDTA-Na2 (372 Da), EGTA (380 Da), 1,10-phenanthroline (198 Da), phosphoramidon (580 Da), Dithiobis (2-amino-4-methylpentane), E-64 (357 Da), cystatin, bestatin, epibestatin hydrochloride, aprotinin, minocycline, ALLN (384 Da), and the like have been used as protein decomposition inhibitors.
Further, the functional chemical substances below may also be added. By adding sodium molybdate, it is possible to stabilize the receptors and thus protect them from decomposition. Glycerol can be used as a protein blocking agent. Dithiothreitol (DTT) has been used as a reducing agent.
Additionally, as buffers, p-toluenesulfonic acid, tartaric acid, citric acid, phthalate, glycine, trans-aconitic acid, formic acid, 3,3-dimethylglutaric acid, phenylacetic acid, sodium acetate, succinic acid, sodium cacodylate, sodium hydrogen maleate, maleic acid, sodium phosphate, KH2PO4, imidazole, 2,4,6-trimethylpyridine, triethanolamine hydrochloride, sodium 5,5-diethylbarbiturate, N-ethylmorpholine, sodium pyrophosphate, tris(hydroxymethyl)aminomethane, bicine, 2-amino-2-methylpropane-1,3-diol, diethanolamine, potassium p-phenolsulfonate, boric acid, sodium borate, ammonia, glycine, Na2CO3/NaHCO3, sodium borate, or a combination of these substances, have been used.
Under such circumstances, selection of additives to be added to the reaction solution is crucially important, and appropriate selection of the additives and optimization of the reaction solution have been desired in the existing bioassays.
In addition, the known bioassays have a problematic need for multiple reaction buffers. This problem has caused the method to become complex, or increased the cost thereof. This problem has also caused the assay steps performed by the user to become complex.
For example, to perform reporter-gene assay, first of all, a plasmid containing a reporter gene is introduced into cultured cells in a microplate. Thereafter, ligand stimulation was performed for about 12 hours. Then, after the medium is discarded, the cells are washed once with a PBS (phosphate buffered saline) buffer (the first buffer). Next, the cells in the microplate are lysed with a cell lysis buffer (the second buffer) for 20 minutes. This cell lysis buffer is mixed with an assay buffer containing a substrate (the third buffer) in a predetermined proportion, and the luminescence value is immediately measured. As such, reporter-gene assay requires at least three buffers and a long measurement step.
Further, in the method using the “integrated-molecule-format bioluminescent probe,” a similar measurement step was necessary. First, an expression vector of the “integrated-molecule-format bioluminescent probe” is introduced into eukaryotic cells cultured in a microplate; the cells are then cultured again for 16 hours. The cells are then subjected to ligand stimulation for 20 to 30 minutes. Finally, the medium is discarded, and the cells are washed once or twice with a PBS buffer (the first buffer). The remaining cells are treated with a cell lysis buffer (the second buffer) for 20 minutes. Thereafter, the lysate is mixed with an assay buffer (the third buffer) containing a substrate at an appropriate ratio, thereby causing a luminescence reaction. The luminescence value is immediately measured with a luminometer (Non-patent Documents 26 and 27).
All of the above known methods use a plurality of reaction buffers, require a cumbersome measurement step, and take a long time. Therefore, there has been a demand for an improved method in which the measurement step can be simplified and the measurement time can be reduced.